The overall objective of this project is to develop a novel chemical class of broad-spectrum therapeutic agents, the bis-indole lead series, for use against Gram-negative category A and B bacterial biodefense threats, and file an IND by the end of the five year grant. The limited number and efficacy of current therapies, increasing drug resistance, and the possibility of deliberate engineering of antibiotic resistant strains argue for the urgent development of new more potent, broad-acting antibacterial agents capable of combating these biodefense threats. Compounds in this new chemical series exhibit broad-spectrum antibacterial activity, displaying potent inhibition (MICs of 0.3-.30ug/ml) of the growth of Gram-negative bacterial species, including a number of Category A and B agents such as Yersinia pestis, Francisella tularensis, Burkholderia mallei and pseudomallei. These compounds are also active against ciprofloxacin-resistant Bacillus anthracis (MIC 0.2-0.4ug/ml) and a variety of other Gram-positive and Gram-negative bacterial species. They act by a rapid bactericidal mechanism, exhibiting 3-log's of killing in 1-4 hours at concentrations near the MIC values. The mechanism of action is unknown. We have not been able to isolate mutants resistant to the primary lead scaffold MBX 1066. The lead series is effective in murine models of Gram-negative infections (ED50 <10 mg/kg). In addition, the compounds are well-tolerated in mice (MTD >400 mg/kg). Finally, these compounds are relatively easy and inexpensive to synthesize, with a very favorable cost of goods. These overall characteristics advocate for the rapid development of these compounds as broad spectrum antibacterial agents for use against biodefense category A and B bacterial pathogens. In this application, we propose to chemically optimize the bis-indole lead series in a rational drug discovery effort focused on improving broad-spectrum potency against gram-negative category A and B bacterial threats, demonstrate efficacy in animal models and establish the mechanism of action. Once a final clinical candidate has been chosen, we will conduct IND-enabling (GLP) pharmacokinetic, toxicology and safety pharmacology studies, in two species, and submit an IND. RELEVANCE (See instructions): Gram-negative category A and B bacterial biodefense pathogens represent a serious threat to the United States. The very limited number and efficacy of current therapies, increasing drug resistance, and the possibility of deliberate engineering of antibiotic resistant strains argue for the urgent development of new more potent, broad-acting antibacterial agents capable of combating these threats. The objective is to develop a new chemical class of broad-spectrum therapeutic agents for use against these threats